Nearly 50 major diseases ranging from diabetes to neurodegenerative disorders including Alzheimer?s (AD), Parkinson?s (PD) and Huntington?s (HD) diseases have been linked to protein misfolding and aggregation. Cells grow and proliferate under the constant threat of damage from endogenous reactive oxygen species (ROS), exogenous oxidants and reactive electrophiles. Cytosolic protein cysteines are almost exclusively maintained in the reduced state, and cysteine oxidation caused by oxidative stress is predicted to result in significant misfolding and aggregation. However, relatively little is known about the consequences of redox imbalance on protein homeostasis (proteostasis). Furthermore, the roles of cellular reduction-oxidation (redox) buffering pathways, including the thioredoxin and glutathione systems, in maintaining cytosolic proteostasis are not well understood. We seek to understand the interplay between cytoprotective stress response pathways and the machinery employed to maintain proteostasis. Published and preliminary results detailed in the proposal lead us to hypothesize that induction of the cytoprotective heat shock response (HSR) by redox imbalance is mediated in part by a cysteine switch in the principal protein chaperone Hsp70 (Ssa1 in budding yeast) and that thiol redox buffering plays a significant role in maintenance of cytosolic proteostasis. The primary objectives of this renewal application are to determine the impacts of thiol-reactive stress on cytoplasmic protein biogenesis and protein quality control and to elucidate the regulatory interactions between oxidant and unfolded protein responses, through three distinct lines of investigation. In Specific Aim 1 we will define the mechanism by which the key molecular chaperone Ssa1/Hsp70 regulates the HSR through modulation of transcriptional activity by the master heat shock transcription factor Hsf1. Specific Aim 2 will investigate the consequences of thiol- reactive stress on Ssa1/Hsp70 activity and cellular functions, including how the Ssa1/Hsp70 redox switch regulates the HSR, and determine the roles of the highly conserved redox buffering systems in mediating thiol-reactive stress. In Specific Aim 3, we will determine the impacts of protein thiol modification on general cytosolic proteostasis using proteomic and genetic approaches. The work outlined in this proposal will reveal the mechanistic connections between cellular redox and protein quality control networks by exploiting the tractable yeast model system. These results in turn will guide future development of therapeutic interventions targeting ROS- and protein quality control-based disorders.